Effective Mechanisms Of Ethanol On Mouse Cerebellar Purkinje Cell Activity And Sensory Information Synaptic Transmission

[Purpose]Cerebellar Purkinje cells (PCs) are sensitive to ethanol, which express a change in simple spike firing rate under in vitro conditions. However, the effective mechanisms of ethanol on spontaneous complex spike (CS) activity of PCs under in vivo conditions are unclear. Therefore, we here investigated the mechanisms of ethanol modulated spontaneous CS activity of PCs in urethane-anesthetized mice by in vivo patch-clamp recording and pharmacological methods.[Methods]Adult (6-8 week-old) ICR mice were anesthetized by intraperitoneal injection of urethane. The mice were tracheotomized and fixed on a custom-made stereotaxic frame. A 1-1.5 mm craniotomy was drilled to expose the cerebellar surface corresponding to Vermis VI-VII. The brain surface was constantly superfused with oxygenated artificial cerebrospinal fluid (ACSF) with a peristaltic pump. Cell-attached and in vivo whole-cell patch-clamp recordings from PCs were performed with an Axopatch-200B amplifier. The signals of PC whole-cell recordings were acquired through a Digidata 1440 series analog-to-digital interface on a personal computer using Clampex 10.3 software. Patch electrodes (4-6 MΩ) contained internal solution.The PCs whole-cell recordings from PCs were performed at depths 150-200μm under pia mater membrane, and identified by regular spontaneous SS accompanied with irregular CS, and confirmed by biocytin histochemistry. Electrophysiological data were analyzed using Clampfit 10.3 software. All values are expressed as the mean ± SEM, and differences were evaluated by the Student’s paired t-test or one-way ANOVA using SPSS 17.0 software. P values below 0.05 were considered to indicate a statistically significant difference between experimental groups.[Results]1.Under current-clamp conditions, cerebellar surface perfusion of ethanol (300 mM) did not significantly change the frequency of SS firing rate, but induced decreases in the CS-evoked pause of SS firing and amplitude of afterhyperpolarization (AHP).2.Under voltage-clamp conditions, application of ethanol significantly depressed the waveform of CS-evoked inward currents, which expressed decreases in the area under curve (AUC) of CS and number of CS spikelet, but without changing frequency and instant frequency of the spontaneous CSs.3.Ethanol-induced decrease in the AUC of spontaneous CS in a concentration-dependent manner. The IC50 of ethanol for decreasing AUC of spontaneous CS was 168.5 mM.4.Blocking N-methyl-D-aspartate receptors (NMDARs) and metabotropic glutamate receptor subtype 1 (mGluR1) induced decreases in number of CS spikelet and area under CS, but failed to prevent ethanol-induced inhibition of CS.5.The ethanol induced inhibition of decreases in the CS-evoked pause of SS firing, amplitude of AHP, number of CS spikelet and area under CS were significant prevented by applying cannabinoids receptor 1 (CB1) antagonist, AM251. Moreover, extracellular application of CB1 receptor agonist, WIN55212-2 significantly induced decreases in number of CS spikelet, AUC, pause of SS and amplitude of AHP.[Conclusions]1. Our present results indicate that ethanol activated cannabinoids CB1 receptor resulting in an inhibition of spontaneous CS activity of PC in vivo in mice, suggesting that excessive ethanol intake inhibits periphery afferent information transferring to cerebellar PC via the activation of presynaptic CB1 receptor at CF-PC synapse.2. The effects of ethanol on CS activity could be in part responsible for the impairments of cerebellar cortical information processing related to acute ethanol intoxication.[Purpose]Acute overdose intake of ethanol can induce a dysfunction of cerebellar motor regulation and cerebellar ataxia. We recently found that ethanol inhibited the sensory stimulation-evoked GABAergic inhibitory responses in the cerebellar cortical molecular layer, suggesting that ethanol modulates the facial stimulation-evoked responses of mouse cerebellar Purkinje cell (PC). Therefore, we here investigated the mechanism of ethanol modulation of facial stimulation-evoked responses of cerebellar PC in urethane-anesthetized mice by in vivo patch-clamp recording and pharmacological methods.[Methods]Adult (6-8 week-old) ICR mice were anesthetized by intraperitoneal injection of urethane. The mice were tracheotomized and fixed on a custom-made stereotaxic frame. A 1-1.5 mm craniotomy was drilled to expose the cerebellar surface corresponding to Vermis VI-VII. The brain surface was constantly supervised with oxygenated ACSF with a peristaltic pump. In vivo whole-cell patch-clamp recordings from PCs were performed with an Axopatch-200B amplifier. The signals of PC whole-cell recordings were acquired through a Digidata 1440 series analog-to-digital interface on a personal computer using Clampex 10.3 software. Patch electrodes (4-6 MΩ) contained internal solution. The PCs whole-cell recordings from PCs were performed at depths 150-200μm under pia mater membrane, and identified by regular spontaneous SS accompanied with irregular CS, and confirmed by biocytin bistochemistry. Facial stimulation was performed by air-puff (30-60 ms,60 psi) of ipsilateral whisker pad by a pressurized injection system. The air-puff stimulations of was controlled by a personal computer, which were synchrinized with the electrophysiologial recordings via a Master 8 controller and Clampex 10.3 software. Electrophysiological data were analyzed using Clampfit 10.3 software. All values are expressed as the mean ± SEM, and differences were evaluated by one-way ANOVA using SPSS 17.0 software. P values below 0.05 were considered to indicate a statistically significant difference between experimental groups.[Results]1. Under current-clamp conditions, air-puff stimulation of ipsilateral whisker-pad evoked inhibitory postsynaptic potential (BPSP) accompanied with a pause of simple spike (SS) firing, which were inhibited by ethanol (300 mM).2. Under voltage-clamp conditions, ethanol (300 mM) inhibited facial stimulation-evoked outward currents which expressed decreases in amplitude, half-width, rise time and decay time of the currents.3. The ethanol-induced inhibition of facial stimulation-evoked outward currents was dose-dependent with the 50% inhibitory concentration (IC50) was 148.5 mM.4. The ethanol induced inhibition of facial stimulation-evoked outward currents was significant prevented by applying cannabinoids receptor 1 (CB1) antagonist, AM251 and O-2050, as well as CB1 receptor agonist, WIN55212-2.[Conclusions]1. Our present results indicate that ethanol inhibits the facial stimulation-evoked outward currents by activation of presynaptic CB1 receptors.2. Overdose intake of ethanol impairs sensory information processing partly by inhibiting of GABA release from MLIs to PC, which provides a novel mechanism of ethanol impairs the cerebellar function.